The present invention relates to attenuated strains of the pathogenic protozoan, Neospora, and to live vaccines against neosporosis prepared from the attenuated strains which are useful in the prevention of clinical disease and abortion in mammals.
Neospora is a pathogenic protozoan parasite of animals which has recently been recognized as a major cause of abortion, neonatal death, congenital infection, and encephalitic disease in mammals. Dubey and Lindsay, 1993, Parasitology Today, 9:452-458. N. caninum infects dogs, and congenitally infects pups, often leading to paralysis. Tachyzoites of N. caninum have been isolated from naturally infected pups. Lindsay and Dubey, 1989, J. Parasitol. 75:163-165. Neospora spp. are a major cause of abortion in dairy cattle. Cases of Neospora related disease, i.e., neosporosis, have also been reported in goats, sheep and horses.
Although N. caninum is superficially similar to the pathogen, Toxoplasma gondii, N. caninum and T. gondii have been distinguished from each other antigenically and ultrastructurally. Dubey and Lindsay, 1993, above. In addition, Neospora-like protozoal parasites isolated from the brains of aborted bovine fetuses and continuously cultured in vitro were shown to be antigenically and ultrastructurally distinct from both T. gondii and Hammondia hammondi, and most similar to N. caninum. Conrad et a/., 1993, Parasitology 106:239-249. Furthermore, analysis of nuclear small subunit ribosomal RNA genes revealed no nucleotide differences between Neospora spp. isolated from cattle and dogs, but showed consistent differences from T. gondii. Marsh et al., 1995, J. Parasitol. 81:530-535.
The etiologic role of a bovine isolate of Neospora in bovine abortion and congenital disease has been confirmed. Barr et al., 1994, J. Vet. Diag. Invest. 6:207-215. A rodent model of central nervous system neosporosis has been developed using inbred BALB/c mice infected with N. caninum. Lindsay et al., 1995, J. Parasitol. 81:313-315. In addition, models to study transplacental transmission of N. caninum in pregnant outbred and inbred mice have been described by Cole et al., 1995, J. Parasitol. 81:730732, and by Long et al., 1996, J. Parasitol. 82:608-611, respectively. Furthermore, an experimental N. caninum pygmy goat model that closely resembles naturally acquired Neospora-induced cattle abortion has been demonstrated. Lindsay et al., 1995, Am. J. Vet. Res. 56:1176-1180.
WO 9525541 discloses a biologically pure culture of bovine Neospora, methods of detecting anti-Neospora antibodies and Neospora-specific nucleic acids, and a composition containing a bovine Neospora antigen and carrier for use as a vaccine. WO 9525541 does not, however, teach attenuated live cultures of Neospora, or live vaccines prepared therefrom which are able to trigger a protective immune response in a vaccinated animal.
In a first aspect, the present invention provides cultures of cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects a mammal against neosporosis when administered as a live vaccine.
In a second aspect, the present invention provides vaccines to protect a mammal against neosporosis, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the mammal against neosporosis when administered as a live vaccine, and a veterinarily acceptable carrier. Vaccines of the invention may further comprise one or more other components including, for example, an adjuvant. Vaccines of the present invention may be administered to any mammalian species susceptible to infection and disease caused by Neospora including, but not limited to, dogs, cows, goats, sheep and horses.
In a third aspect, the present invention provides methods for preparing cultures of attenuated cells from a pathogenic strain of Neospora for use in a vaccine that protects a mammal against neosporosis, comprising modifying cells from a pathogenic parent strain of a species of Neospora; selecting and clonally propagating one or more modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain; and selecting and clonally propagating one or more attenuated cells which are capable of triggering an immune response that protects the mammal against neosporosis when administered in a live vaccine.
In a fourth aspect, the present invention provides methods for preparing a vaccine that protects a mammal against neosporosis, comprising modifying cells from a pathogenic parent strain of a species of Neospora; selecting and clonally propagating those modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain but which are capable of triggering an immune response in the mammal that protects against neosporosis when administered in a live vaccine; and combining an immunologically effective amount of the attenuated cells with a veterinarily acceptable carrier in a form suitable for administration as a live vaccine to the mammal.
In a fifth aspect, the present invention provides methods for vaccinating a mammal against neosporosis, comprising administering to the mammal an immunologically effective amount of a vaccine comprising live cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the mammal against neosporosis when administered as a live vaccine, and a veterinarily acceptable carrier.
In a sixth aspect, the present invention provides combination vaccines, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the mammal against neosporosis when administered as a live vaccine; one or more other antigens that trigger an immune response that protects the mammal against a disease or a pathological condition; and a veterinarily acceptable carrier. The combination vaccines may further comprise one or more other components including, for example, an adjuvant.
Applicants have discovered that cells of a pathogenic strain of a species of Neospora may be attenuated, and that the resulting attenuated cells are capable of triggering an immune response that protects mammals against neosporosis when administered as a live vaccine. The present invention thus provides cultures of cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects a mammal against neosporosis when administered as a live vaccine.
The present invention further provides methods for preparing cultures of attenuated cells of a species of Neospora for use in a vaccine that protects a mammal against neosporosis, comprising modifying cells from a pathogenic parent strain of a species of Neospora, for example, by high serial passage, or by exposure to a mutagenic agent, or by genetic engineering using recombinant DNA techniques; selecting and clonally propagating one or more modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain; and selecting and clonally propagating one or more attenuated cells which are capable of triggering an immune response that protects the mammal against neosporosis when administered in a live vaccine.
As used herein, the term xe2x80x9cneosporosisxe2x80x9d refers to infection of a mammal by a species or strain of Neospora, or to any clinical symptom, condition, event or pathology associated with infection of the mammal by Neospora.
The term xe2x80x9cattenuatedxe2x80x9d as used herein describes a cell, culture, or strain of Neospora exhibiting a detectable reduction in infectivity or virulence in vitro or in vivo as compared to that of the parent strain of Neospora from which the attenuated cell, culture, or strain is derived. Reduction in virulence encompasses any detectable decrease in any attribute of virulence, including infectivity in vitro or in vivo, or any decrease in the severity or rate of progression of any clinical symptom or condition associated with infection.
The term xe2x80x9cparent strainxe2x80x9d refers to a strain of Neospora which exhibits a relatively higher degree of pathogenicity when administered to a mammal than an attenuated strain which is derived therefrom by one or more passages in vivo or in vitro and/or one or more attenuation steps.
The present invention further encompasses preparation and use in a vaccine of cells of a strain of Neospora derived from a strain or species that is not pathogenic in a particular mammalian species, but which cells have been modified by chemical or genetic means to be capable of triggering a protective immune response in members of that mammalian species.
The live attenuated cells of the invention are capable of triggering an immune response that protects a mammal against neosporosis after one or more administrations as a live vaccine. A xe2x80x9cprotective immune responsexe2x80x9d is defined as any immunological response, either antibody or cell mediated immunity, or both, occurring in the mammal that either prevents or detectably reduces subsequent infection, or eliminates or detectably reduces the severity, or detectably slows the rate of progression, of one or more clinical symptoms or conditions associated with neosporosis. The term xe2x80x9cimmunologically effective amountxe2x80x9d refers to that amount or dose of vaccine or antigen that triggers a protective immune response when administered to a mammal.
Preparation of Attenuated Strains of Neospora
Since the invention is based on the discovery that cells of a pathogenic strain of Neospora may be attenuated, and that the resulting attenuated cells are capable of triggering an immune response that protects a mammal against neosporosis when administered as a live vaccine, practice of the invention is not limited to any particular method of attenuation. Rather, attenuation of cells of a pathogenic strain of Neospora may be carried out by any techniques or procedures known in the art including, but not limited to, high serial passage, or exposure to a mutagenic agent, or by genetic engineering using recombinant DNA technology, or some combination thereof.
High serial passage may be carried out by repeated in vitro passaging of cells of a pathogenic strain of Neospora in susceptible host cells until sufficient attenuation occurs. Passaging may be conducted under specific environmental conditions to select for attenuated cells. For example, passaging may be conducted at a temperature below that of the body temperature of the intended mammalian vaccinate to select for temperature-sensitive strains of Neospora that will not grow, or that will only grow at a reduced rate, when administered in a vaccine to the mammal.
Mutagenesis may be carried out by exposure of Neospora cells to either a chemical mutagen or to radiation, as described in the art. A non-limiting example of a chemical mutagen useful in the practice of the invention is N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (MNNG) (Sigma), the use of which is described below in Example 1. Radiation may be selected from either ultraviolet light or ionizing radiation.
The degree of exposure to the mutagen, ie., the concentration of chemical mutagen, or the level of radiation, as well as the duration of exposure, is preferably that amount which results in producing one or more viable cells of Neospora that exhibit an attenuated level of pathogenicity but that are capable of triggering an immune response that protects against neosporosis when administered as a live vaccine to a mammal Appropriate parameters for use of mutagenic agents may be determined empirically using standard techniques.
Pathogenic strains of Neospora may also be attenuated using recombinant DNA technology according to techniques known in the art, and the present invention is intended to encompass such modified strains and vaccines prepared therefrom. Non-limiting examples of recombinant DNA techniques which may be used to practice the invention include gene replacement or gene knockout to disable one or more genes, resulting in a strain having an attenuated pathogenicity. Genes that may be disabled include, for example, an essential metabolic gene, or a gene encoding a virulence factor, or a gene encoding a surface antigen that plays a role in modulating the immune response in the mammalian host.
A non-limiting example of an essential metabolic gene that may usefully be targeted for disruption in the Neospora genome is the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Titus et al., 1995, Proc. Natl. Acad. Sci. USA, 92:10267-10271, describe knocking out the DHFR-TS gene to produce a safe, live Leishmania vaccine, which publication is incorporated by reference. By disrupting the DHFR-TS gene in Neospora, auxotrophic mutants will be created that require thymidine for continued growth, that exhibit attenuated pathogenicity, and that are capable of triggering an immune response in a mammal that protects against neosporosis when administered as a live vaccine.
Recombinant DNA techniques for gene replacement or gene knockout are known in the art and include, but are not limited to, those that take advantage of homologous recombination. For example, cells of a pathogenic strain of Neospora may be transformed or transfected with a vector, such as a plasmid, comprising homologous nucleotide sequences that normally flank, or are located within, for example, an essential metabolic gene, preferably a single copy gene, in a pathogenic strain of Neospora. Between or within the homologous nucleotide sequences, the vector may further comprise a nucleotide sequence that corresponds to the nucleotide sequence in the pathogenic strain but which is defective as a result, for example, of a xe2x80x9cnon-silentxe2x80x9d change or deletion in one or more nucleotides compared to the sequence from the pathogenic strain. Transformation of a cell of the pathogenic strain with the vector is followed by integration of the defective gene sequence into the Neospora genome, which also serves to replace the original or xe2x80x9cwild-typexe2x80x9d sequence. Thus, the targeted gene is disabled in the transformed cell. Transformed cells may then be screened for those cells that exhibit an attenuated pathogenicity. Transformed cells exhibiting attenuated pathogenicity may then be screened again for those cells that are capable of triggering an immune response in a mammal that protects against neosporosis when administered as a live vaccine.
To aid in the selection of transformants, the vector may be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker. Reporter genes which may be useful in the invention are well-known in the art and include, for example, the gene encoding chloramphenicol acetyl transferase (CAT), or the gene encoding luciferase. A further non-limiting example of a reporter gene is a sequence encoding E. coli xcex2-galactosidase, which can be inserted into the vector and used to confirm transformants by detecting enzymatic activity through conversion of a substrate such as, for example, red-xcex2-D-galactopyranoside, to a colored product. Seeber and Boothroyd, 1996, Gene, 169:39-45, used this reporter enzyme to detect transformants in Toxoplasma gondii, which publication is incorporated herein by reference.
Coding sequences that encode selectable markers which may be useful in the invention are also well-known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement Examples of such sequences include those that confer resistance to hygromycin, or to neomycin, or to phleomycin. An example of the use of an antibiotic resistance marker in a different pathogen is presented by Messina et al., 1995, Gene, 165:213-217, which describes the use of a resistance marker for phleomycin to construct stable transformants in T. gondii, and which publication is incorporated herein by reference.
Any coding sequence for a reporter gene product or selectable marker should preferably be inserted into the vector in operative association with a regulatory element coding sequence. As used herein, a xe2x80x9cregulatory elementxe2x80x9d includes, but is not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression Also, as used herein, a DNA coding sequence is in xe2x80x9coperative associationxe2x80x9d with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the DNA coding sequence and/or the translation of the corresponding mRNA. For example, vectors for expression of the selectable marker ble in Neospora cells may be constructed by flanking the ble open reading frame (ORF) with regulatory regions from genes of Neospora or from the closely related Apicomplexa, Toxoplasma. The 5xe2x80x2 flanking regions from different single copy Neospora or Toxoplasma genes may be used to express ble. Examples of single copy Toxoplasma genes are: (i) SAG1, encoding the major tachyzoite surface antigen, p30 (Burg et al., 1988, J. Immunol. 141:3584-3591); (ii) GRA1, encoding a secretory protein, p23 (Cesbron-Delauw et al., 1989, Proc. Natl. Acad. Sci. 86:7537-7541); and (iii) GRA2, encoding a secretory protein, p28 (Mercier et al., 1993, Mol. Biochem. Parasitol. 58:71-82). Examples of single copy Neospora genes include: (i) homologs of the Toxoplasma SAG1, GRA1 and GRA2 genes, identified using standard PCR methods based, for example, on the published Toxoplasma sequences; (ii) the gene encoding the major cell surface protein, NC-p43, of N. caninum tachyzoites (Hemphill, 1996, Infect. Immun. 64:4279-4287); and (iii) genes encoding the immunodominant 17-, 29-, 30- and 37 kDa excretory/secretory proteins (Bjerkas et al., 1994, Clin. Diag. Lab. Immun. 1:214-221). The 3xe2x80x2 flanking region from the SAG1 gene may be used to provide a polyadenylation sequence. The vector backbone for insertion of the 5xe2x80x2 promoter, ble gene, and 3xe2x80x2 polyadenylation sequences may be any standard, commercially available plasmid, such as pBLUESCRIP(trademark) (Stratagene).
Once an appropriate vector is constructed, it is used to transform or transfect one or more cells from a parental strain of Neospora. The vector may be introduced into the cells in accordance with known techniques, including but not limited to electroporation, microinjection, viral transfection, liposome-mediated transfection, microprojectile bombardment, etc.
Once the vector is introduced into the Neospora cells, the presence, integration and maintenance of the introduced coding sequence into the host cell genome, or episomally, can be confirmed and monitored by standard techniques including, but not limited to, Southern hybridization analysis; PCR analysis, including reverse transcriptase-PCR (RT-PCR); immunological or colorimetric assay for the expected protein product; detecting the presence or absence of a marker gene function, such as appearance of a novel auxotrophy; or by detecting an attenuation in pathogenicity.
Examples of vector construction, transformation, selection of transformants, host cell expression, etc., as applied specifically to pathogenic protozoans, are described in the following publications, which are incorporated herein by reference: Seeber and Boothroyd, 1996, above; Titus et al., 1995, above; Messina et al., 1995, Gene, 165:213-217; Sibley et al., 1994, Proc. Natl. Acad. Sci. USA, 91:5508-5512; Donald and Roos, 1994, Mol. Biochem. Parasitol., 63:243-253; Kim et al., 1993, Science, 262:911-914; Ryan et al., 1993, Proc. Natl. Acad. Sci. USA, 90:8609-8613; Soldati and Boothroyd, 1993, Science, 260:349-352; Eid and Sollner-Webb, 1991, Proc. Natl. Acad. Sci. USA, 88:2118-2121; LeBowitz et al., 1990, Proc. Natl. Acad. Sci. USA, 87:9736-9740; Lee and Van der Ploeg, 1990, Science, 250:1583-1586; Asbroek et al., 1990, Nature, 348:174-175; Cruz and Beverley, 1990, Nature, 348:171-173; and Laban et al., Nature, 343:572-574.
General techniques of genetic recombination, including vector construction, transformation, selection of transformants, host cell expression, etc., are further described in Maniatis et al., 1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.; Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego, Calif.; and Erlich (ed), 1992, PCR Technology, Oxford University Press, New York, which are incorporated herein by reference.
After the attenuation step, cells that exhibit one or more indicators of attenuated pathogenicity are selected from the culture and clonally propagated after limiting dilution. Examples of such indicators include, but are not limited to, appearance of a novel temperature-sensitivity or a novel auxotrophy in vitro, or a reduction in a virulence attribute such as infectivity or severity or rate of progression of one or more symptoms or conditions in a mammal after administration of cells of the strain as compared to infection with the parent strain, among others. A particular, non-limiting example of a temperature-sensitivity that is useful in practicing the invention is one in which cells of the attenuated strain will grow at 32xc2x0 C., but not at 37xc2x0 C. Such a temperature-sensitive strain will cause the lysis of infected host cells at 32xc2x0 C., resulting in the appearance of lesions or plaques in a host cell monolayer. When grown at 37xc2x0 C., the attenuated strain will not replicate sufficiently and will thus fail to produce plaques in host cell monolayers.
An attenuated strain of Neospora may be derived from any pathogenic strain of any species of the genus including, but not limited to, N. caninum. A non-limiting example of a particular pathogenic strain of N. caninum from which an attenuated strain may usefully be derived is strain NC-1 which is present in infected MARC145 monkey kidney cells from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 USA (ATCC Accession No. CRL-12231). Strain NC-1 is also described in Dubey et al., 1988, J. Am. Vet. Med. Assoc. 193:1259-63, which publication is incorporated herein by reference. Alternatively, pathogenic strains of Neospora may be obtained from tissues, organs or body fluids of infected animals exhibiting clinical symptoms of neosporosis using standard isolation techniques described, for example, in the publications reviewed above. A non-limiting example of a live, attenuated strain derived from the NC-1 strain of N. caninum is NCTS-8 which is present in infected MARC145 monkey kidney cells from the ATCC (ATCC Accession No. CRL-12230).
Both parental strains and attenuated strains of Neospora may be cultured in vitro by infecting any receptive cell line, preferably a mammalian cell line, with tachyzoites of the strain according to known techniques described in the art. Mammalian cell lines in which tachyzoites of Neospora can be cultured include, for example, human foreskin fibroblasts (Lindsay et al., 1993, Am. J. Vet. Res. 54:103-106); bovine cardiopulmonary aortic endothelial cells (Marsh et al., 1995, above); and bovine monocytes (Lindsay and Dubey, 1989, above), among others. For example, tachyzoites of N. caninum may be cultured in monolayers of Hs68 human foreskin fibroblast cells (ATCC Accession No. CRL-1635) (Lindsay et al., 1993, above). Bradyzoites may be similarly cultured and manipulated.
Mammalian cell cultures can be grown, and cell cultures infected with Neospora can be maintained, in any one of several culture media described in the art. For example, stationary monolayer cultures of bovine cardiopulmonary aortic endothelial cells infected with tachyzoites of N. caninum may be grown in Dulbecco""s Minimum Essential Medium (DMEM: Gibco Laboratories, N.Y.), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) or adult equine serum (ES), 2 mM L-glutamine, 50 U/ml penicillin, and 50 xcexcg/ml streptomycin (Conrad et al., 1993, above). Monolayers of Hs68 human foreskin fibroblast cells may be maintained in RPMI 1640 (Gibco) containing 2% (v/v) fetal bovine serum, 1.0 mM sodium pyruvate, 1xc3x97104 U/ml penicillin, 1xc3x97104 xcexcg/ml streptomycin, 5xc3x9710xe2x88x922 mM 2-mercaptoethanol and 0.3 mg/ml L-glutamine (maintenance medium). Monolayer cultures of Hs68 human foreskin fibroblast cells infected with Neospora may be maintained in identical media, but in which the fetal bovine serum is increased to 10% (v/v) (growth medium). Attenuated strains of Neospora having novel auxotrophies will require appropriate modification to the culture medium to support growth, as known in the art.
Neospora-infected monolayer cultures of mammalian cells are typically maintained under standard tissue culture conditions such as, for example, at 37xc2x0 C. and 5% CO2. Tachyzoites are generally passaged to uninfected monolayer cultures when 70-90% of the mammalian cells in the culture have become infected, which may be determined microscopically using standard techniques. Tachyzoites may be collected from the infected mammalian cell cultures by lysing the host cells using any standard technique that allows the tachyzoites to retain viability, and collecting the tachyzoites by filtration or by centrifugation, for example.
Preparation and Use of Vaccines
The present invention provides vaccines against neosporosis, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the mammal against neosporosis when administered as a live vaccine, and a veterinarily acceptable carrier.
The present invention further provides methods for preparing a vaccine that protects a mammal against neosporosis, comprising modifying cells from a pathogenic parent strain of a species of Neospora; selecting and clonally propagating those modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain but which are capable of triggering an immune response in the mammal that protects against neosporosis when administered in a live vaccine; and combining an immunologically effective amount of the attenuated cells with a veterinarily acceptable carrier in a form suitable for administration as a live vaccine to the mammal.
The present invention further provides methods of vaccinating a mammal against neosporosis, comprising administering to the mammal an immunologically effective amount of a vaccine comprising live cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the mammal against neosporosis when administered as a live vaccine, and a veterinarily acceptable carrier.
The vaccine of the invention comprises live cells of an attenuated strain of Neospora, either as the sole antigenic component or in combination with one or more other antigens that trigger an immune response that protects a mammal against a disease or pathological condition which may or may not be related to neosporosis. Thus, the present invention further provides combination vaccines, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the mammal against neosporosis when administered as a live vaccine; one or more other antigens that trigger an immune response that protects the mammal against a disease or a pathological condition; and a veterinarily acceptable carrier. The combination vaccines may further comprise one or more other components including, for example, an adjuvant.
The vaccine is conventionally administered parenterally, for example, either by subcutaneous or intramuscular injection. However, the vaccine may also be administered by intraperitoneal or intravenous injection, or by other routes, including orally, intransally, rectally or vaginally, and where the vaccine is so administered, a veterinarily acceptable carrier is appropriately selected. The vaccine may simply comprise attenuated cells in culture fluid, which are administered directly to the mammal. Alternatively, the vaccine may comprise attenuated cells combined with a veterinarily or pharmaceutically acceptable carrier selected from those known in the art based on the route of administration and its ability to maintain cell viability. Non-limiting examples of such carriers include water, saline, buffered vehicles and the like. Suitable other vaccine vehicles and additives are known, or will be apparent, to those skilled in the art. See, e.g., Remington""s Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
The vaccine may further comprise one or more other components such as an immunomodulatory agent including, for example, interleukin-1, or another immuno-enhancing substance such as a veterinarily acceptable adjuvant. Non-limiting examples of adjuvants include Freund""s complete and incomplete adjuvants, mineral gels including, for example, aluminum hydroxide, and oil-in-water or water-in-oil formulations. Immunomodulatory agents are selected based on their ability to maintain both viability of the attenuated Neospora cells and ability of the cells to trigger a protective immune response in the vaccinated mammal.
Non-limiting examples of oil-in-water formulations that are useful as adjuvants in the vaccines of the invention include emulsions 1-3, as follows. Emulsion 1 consists of: (a) ME6201 (5% v/v squalene, 0.1% v/v vitamin E, and 0.8% v/v Tween(trademark) 80 dispersant); (b) Quil A (QA) saponin preparation (Superfos) (200 xcexcg/ml); and (c) cholesterol (chol.) (100 xcexcg/ml). Emulsion 2 consists of: (a) ME6201; and (b) Avridine lipoidal amine (1 mg/ml). Emulsion 3 consists of ME6210 (5% v/v squalene, 1.0% v/v vitamin E, and 0.8% v/v Tween(trademark) 80 dispersant).
An effective dosage may be determined by conventional means, starting with a low dose of attenuated cells and then increasing the dosage while monitoring the effects, and systematically varying the dosage as well. Numerous factors may be taken into consideration when determining an optimal dosage per animal. Primary among these is the species, the size of the animal, the age of the animal, the general condition of the animal, the presence of other drugs in the animal, the virulence of a particular strain of Neospora against which the animal is being vaccinated, and the like. The actual dose would preferably be chosen after consideration of the results of other animal studies.
Vaccine regimens are selected also based on the above-described factors. Animals may be vaccinated at any time, including just prior to or at the time of breeding. Supplemental administrations, or boosters, may be required for full protection. One method of detecting whether adequate immune protection has been achieved is to determine seroconversion and antibody titers in the animal after vaccination. Thus, the vaccine of the invention may be administered at any time during the life of a particular animal to be vaccinated, depending upon several factors, including, for example, the timing of an outbreak of neosporosis among other animals, etc. The vaccine may be administered to animals of weaning age or younger, or to more mature animals, for example, as a pre-breeding vaccine to protect against Neospora-related congenital disease or abortion. Effective vaccination may require only a primary vaccination, or a primary vaccination with one or more booster vaccinations. Booster vaccinations may be administered at any time after primary vaccination depending, for example, on the immune response after primary vaccination, the severity of the outbreak, the virulence of the strain of Neospora causing the outbreak, the health of the vaccinate, etc. The timing of vaccination and the number of boosters, if any, will preferably be determined by a veterinarian based on analysis of all relevant factors, some of which are described above.
The Neospora cells used in the vaccine are preferably tachyzoites, but may instead be bradyzoites, or oocysts, or some combination thereof. The concentration of attenuated cells in the vaccine preferably ranges from about 1xc3x97103/ml to about 1xc3x97108/ml, and more preferably from about 2xc3x97106/ml to about 2xc3x97107/ml. A suitable dosage size ranges from about 0.5 ml to about 1.0 ml. Generally, on a per-dose basis, the number of attenuated cells administered to an animal is preferably from about 2xc3x97104 to about 2xc3x97108; more preferably from about 2xc3x97105 to about 2xc3x97107; and most preferably from about 1xc3x97106 to about 1xc3x97107.
The vaccine of the invention protects a mammal against infection or disease caused by Neospora. The vaccine is useful to protect both pregnant and non-pregnant mammals including, but not limited to, bovine, ovine, caprine, canine and equine species, against infection, clinical disease or abortion resulting from neosporosis. The term xe2x80x9cprotectionxe2x80x9d is used broadly and is not limited to absolute prevention of infection by Neospora, but includes a reduction in infectivity, or in the severity of a disease or condition resulting from infection, including a detectable reduction in one or more of the pathological effects or symptoms resulting from infection, or a detectable reduction in the rate of progression of one or more of such pathological effects or symptoms. The vaccine of the invention is also safe, i.e., it does not cause disease or significant side effects in the vaccinated mammal.
The following examples are offered to further illustrate, but not limit, the compositions and methods of the invention.